RESUMO
Recent data suggest that mammary carcinogenesis may be driven by cancer stem cells (CSCs) derived from mutated adult stem cells, which have acquired aberrant cell self-renewal or by progenitor cells that have acquired the capacity for cell self-renewal. Spontaneous mammary cancers in cats and dogs are important models for the understanding of human breast cancer and may represent alternative species model systems that can significantly contribute to the study of human oncogenesis. With the goal of identifying markers for isolating human breast CSCs, we have generated a canine model system to isolate and characterize normal and CSCs from dog mammary gland. Insight into the hierarchical organization of canine tumours may contribute to the development of universal concepts in oncogenesis by CSCs. Cells with stem cell properties were isolated from normal and tumoural canine breast tissue and propagated as mammospheres and tumourspheres in long-term non-adherent culture conditions. We showed that cells obtained from spheres that display self-renewing properties, have multi-lineage differentiation potential, could generate complex branched tubular structures in vitro and form tumours in NOD/SCID mice. We analysed these cells for the expression of human stem and CSC markers and are currently investigating the tumour-initiating properties of these cells and the hierarchical organization of normal and neoplastic canine mammary tissue.
Assuntos
Neoplasias Mamárias Animais , Células-Tronco Neoplásicas/citologia , Animais , Biomarcadores Tumorais , Doenças do Cão/fisiopatologia , Cães , Feminino , Neoplasias Mamárias Experimentais , Camundongos , Camundongos Endogâmicos NOD , Células Tumorais CultivadasRESUMO
Tumors derived from rat LA7 cancer stem cells (CSCs) contain a hierarchy of cells with different capacities to generate self-renewing spheres and tubules serially ex vivo and to evoke tumors in vivo. We isolated two morphologically distinct cell types with distinct tumorigenic potential from LA7-evoked tumors: cells with polygonal morphology that are characterized by expression of p21/(WAF1) and p63 and display hallmarks of CSCs and elongated epithelial cells, which generate tumors with far less heterogeneity than LA7 CSCs. Serial transplantation of elongated epithelial cells results in progressive loss of tumorigenic potential; tumor heterogeneity; CD44, E-cadherin, and epithelial cytokeratin expression and increased alpha-smooth muscle actin I and vimentin expression. In contrast, serial transplantation of LA7 CSCs can be performed indefinitely and results in tumors that maintain their heterogeneity, consistent with self-renewal and multilineage differentiation potential. Collectively, our data show that polygonal cells are CSCs, whereas epithelial elongated cells are lineage-committed progenitors with tumorigenic potential, and suggest that tumor progenitors, although lacking indefinite self-renewal potential, nevertheless may make a substantial contribution to tumor development. Because LA7 cells can switch between conditions that favor maintenance of pure CSCs vs. differentiation into other tumor cell types, this cell system provides the opportunity to study factors that influence CSC self-renewal and differentiation. One factor, p63, was identified as a key gene regulating the transition between CSCs and early progenitor cells.
Assuntos
Glândulas Mamárias Animais/citologia , Neoplasias Mamárias Experimentais/patologia , Células-Tronco Neoplásicas/citologia , Animais , Diferenciação Celular , Linhagem Celular Tumoral , Linhagem da Célula , Células Clonais , Modelos Animais de Doenças , Feminino , Imuno-Histoquímica , Glândulas Mamárias Animais/metabolismo , Camundongos , Camundongos SCID , Neoplasias/metabolismo , Neoplasias/patologia , Células-Tronco Neoplásicas/metabolismo , Ratos , Células-Tronco/citologiaRESUMO
The COOH-terminal fragment of procollagen type I (C3) is produced in tissues with high synthesis of collagen I, such as in breast cancer stroma and in bone. We previously demonstrated that C3 is chemoattractant for breast carcinoma and endothelial cells, and that in tumor cells it induces expression and activation of metalloproteinases (MMP) -2 and -9. Here we demonstrate that C3 induces expression of vascular-endothelial growth factor (VEGF) and of CXCR4, the receptor of the CXCL12/SDF-1 chemokine, in MDA MB 231 breast cancer cells. We show that the changes in gene expression and motility induced by C3 occur in a timely succession and are mediated by multiple and different signaling pathways. C3 induces early phosphorylation of p38/MAPK. Induction of VEGF expression requires continual activity of p38/MAPK and of Protein Kinase C (PKC). Pro-MMP-2 and -9 are induced through a signaling pathway involving G0alpha.i protein, and cell migration requires the activity of a combination of these signaling pathways. Our results suggest that C3 acts as a stromal-derived, cancer-promoting agent active in inducing the migratory phenotype and the survival of cancer cells and determining timely changes in their gene expression that establish conditions promoting tumor angiogenesis and invasion.
Assuntos
Neoplasias da Mama/metabolismo , Colágeno Tipo I/metabolismo , Fragmentos de Peptídeos/metabolismo , Pró-Colágeno/metabolismo , Receptores CXCR4/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Quimiotaxia/fisiologia , Colágeno Tipo I/genética , Feminino , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/genética , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Humanos , Metaloproteinases da Matriz/genética , Metaloproteinases da Matriz/metabolismo , Neuropilina-1/genética , Neuropilina-1/metabolismo , Fragmentos de Peptídeos/genética , Pró-Colágeno/genética , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/metabolismo , Receptores CXCR4/genética , Receptores de Fatores de Crescimento do Endotélio Vascular/genética , Receptores de Fatores de Crescimento do Endotélio Vascular/metabolismo , Sistemas do Segundo Mensageiro/fisiologia , Fator A de Crescimento do Endotélio Vascular/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Quinases da Família src/genética , Quinases da Família src/metabolismoRESUMO
Background: Language deficits cause difficulties in the family, school and social settings, so early detection and intervention are crucial. In Primary Care, children undergo developmental screening using the TEPSI test, which includes language at 4 years-old. Objective: Establish the frequency of language delay in children at pre-school, determined by specific language tests, in order to establish their concordance with TEPSI test. Method: Children between 3 and 5 years-old, attending 2 low-income pre-school facilities from the North Metropolitan Area, were evaluated during 2006. The information was obtained in a blind and simultaneous mode through TEPSI test performed by nurses and 3 specific language tests performed by speech therapists. A performance < p10 or < 2SD in one or was more language tests was considered a deficit. The concordance and discordance between both evaluations were established. Results: From a total of 219 children, 194 (89 percent) completed the evaluation. 48 percent had a language deficit by speech evaluation and 13,9 percent by TEPSI test. The concordance between both evaluations was poor (Kappa 0,2), with a significant discordance (p < 0,0000) by Mc NemarÆs X². Conclusion: The frequency of language problems in this population is high; a poor concordance between the tests used in Primary Care and language evaluations performed by speech therapists was found. The differences could be caused by the different aspects of language being evaluated. The findings lead to reconsider the screening strategies used in Primary Care and to implement language stimulation programs directed to low-income populations at high risk for language deficits.
Introducción: Los déficit del lenguaje conllevan dificultades en el contexto familiar, escolar y social, siendo fundamental la pesquisa e intervención precoz. En la atención primaria (APS) el lenguaje se evalúa en el marco del desarrollo psicomotor (DSM), a los 4 años mediante el test de TEPSI. Objetivo: Describir la frecuencia de déficit de lenguaje en preescolares asistentes a jardín infantil según pruebas de lenguaje específicas y establecer la concordancia entre estas pruebas y el TEPSI. Metodología: Se evaluó a todos los niños entre 3 y 5 años, asistentes a dos jardines infantiles de nivel socioeconómico bajo, del área Norte de la Región Metropolitana. La información se obtuvo en forma simultánea y ciega mediante la aplicación del TEPSI por enfermeras y la evaluación del lenguaje con dos pruebas específicas aplicadas por fonoaudiólogos. Se consideró un desempeño deficitario cuando el rendimiento en una o más pruebas de lenguaje fue < p10 ó < 2DS para la edad. Se estableció la concordancia y discordancia entre ambas evaluaciones. Resultados: De un total de 219 niños, 194 (89 por ciento) completaron la evaluación con los instrumentos señalados. 48,8 por ciento presentó dificultades del lenguaje según la evaluación fonoaudiológica y 13,9 por ciento según TEPSI. La concordancia entre ambas evaluaciones fue pobre (Kappa 0,2), con una discordancia altamente significativa p < 0,0000 según X² de Mc Nemar. Conclusión: Destaca la alta frecuencia de problemas de lenguaje en la población estudiada y la pobre concordancia entre las pruebas aplicadas en APS y la evaluación fonoaudiológica. Las diferencias podrían deberse a los distintos aspectos del lenguaje considerados en las evaluaciones. Los hallazgos invitan a replantear las estrategias de pesquisa utilizadas en APS y a la implementación de programas integrales de estimulación en poblaciones desfavorecidas, consideradas de riesgo para problemas de lenguaje.
Assuntos
Humanos , Masculino , Feminino , Pré-Escolar , Testes de Linguagem , Peneiramento de Líquidos , Transtornos do Desenvolvimento da Linguagem/diagnóstico , Transtornos do Desenvolvimento da Linguagem/epidemiologia , Transtornos do Desenvolvimento da Linguagem/prevenção & controle , Chile , Estudos Transversais , Fatores Socioeconômicos , Área UrbanaRESUMO
The cancer stem cell hypothesis posits that tumors are derived from a single cancer-initiating cell with stem cell properties. The task of identifying and characterizing a single cancer-initiating cell with stem cell properties has proven technically difficult because of the scarcity of the cancer stem cells in the tissue of origin and the lack of specific markers for cancer stem cells. Here we show that a single LA7 cell derived from rat mammary adenocarcinoma has the following properties: the differentiation potential to generate all of the cell lineages of the mammary gland; the ability to generate branched duct-like structures that recapitulate morphologically and functionally the ductal-alveolar-like architecture of the mammary tree; and the capacity to initiate heterogeneous tumors in nonobese diabetic-SCID mice. In addition, we show that cultured cells derived from tumors generated by a single LA7 cell-injection have properties similar to LA7 cells, can generate all of the cell lineages of the mammary gland, and recapitulate the ductal-alveolar-like architecture of the mammary tree. The properties of self-renewal, extensive capacity for proliferation, multilineage differentiation potential, and single-cell tumor-initiation potential suggest that LA7 cells are cancer stem cells and can be used as a model system to study the dynamics of tumor formation at the single-cell level.
Assuntos
Diferenciação Celular , Proliferação de Células , Células-Tronco Neoplásicas/patologia , Adenocarcinoma/patologia , Animais , Benzimidazóis/metabolismo , Neoplasias da Mama/patologia , Carbazóis/metabolismo , Linhagem Celular Tumoral , Linhagem da Célula , Células Cultivadas , Células Clonais , Feminino , Técnica Indireta de Fluorescência para Anticorpo , Corantes Fluorescentes/metabolismo , Imuno-Histoquímica , Queratina-14/metabolismo , Queratina-18/metabolismo , Glândulas Mamárias Animais/patologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Células-Tronco Neoplásicas/metabolismo , Técnicas de Cultura de Órgãos , Ratos , Transplante de Células-Tronco , Transplante HeterólogoRESUMO
Newborn gene therapy, because it can prevent the damage caused by the onset of a disease, deserves specific attention. To evaluate gene transfer in tissues of newborn mice, we used a human immunodeficiency virus (HIV)-2 based lentiviral vector pseudotyped with vesicular stomatitis virus G glycoprotein expressing the green fluorescent protein reporter gene under the control of the cytomegalovirus promoter. We found that very low doses of HIV-2 could infect and be expressed in newborn mice. Under these conditions, the virus was preferentially expressed in the liver and hepatocytes were the predominant target. The treatment was not toxic, the infected liver cells proliferated and the transduced gene was stably expressed. Adult mice could be infected by HIV-2, but the vector was detected in the liver only utilizing the sensitive method of polymerase chain reaction coupled with Southern blot. Direct comparison between newborn and adult recipients demonstrated a much greater efficiency of liver transduction in the newborn mouse. These results indicate that the combination of early intervention and low multiplicity of infection may be a strategy for preferentially and efficiently targeting newborn liver for gene therapy applications.
Assuntos
Animais Recém-Nascidos , Terapia Genética/métodos , Vetores Genéticos/administração & dosagem , HIV-2/genética , Hepatócitos/metabolismo , Transdução Genética/métodos , Adenoviridae/genética , Animais , Southern Blotting , Proliferação de Células , Feminino , Expressão Gênica , Proteínas de Fluorescência Verde/análise , Proteínas de Fluorescência Verde/genética , Hepatócitos/citologia , Fígado/metabolismo , Pulmão/metabolismo , Camundongos , Camundongos Endogâmicos , Microscopia de Fluorescência , Reação em Cadeia da Polimerase/métodos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Baço/metabolismoRESUMO
The study of the development of the mammary gland at the molecular level in the animal is difficult because of the complex tissue organization of the gland. We have previously developed an in vitro system for genetic analysis of mammary cell differentiation, based on the cell line LA7 clonally derived from a rat mammary adenocarcinoma. This cell line, after induction with DMSO, differentiates forming structures called domes. This process is under strict gene regulation, and we have previously identified several of the genes involved. In the present paper, we have defined the meaning of dome formation in relation to mammary development, by showing that treatment of LA7 cells with the lactogenic hormones hydrocortisone and prolactin induces dome formation; in the animal, these hormones precede and accompany milk production. Moreover, dome formation is accompanied by expression within the cells of the milk protein genes WDMN1 and beta-casein, which are differentiation markers for the gland during pregnancy and lactation. We also show that two proteins, highly expressed in the mammary gland during lactation, HSP90-beta and annexin I, are strongly expressed in DMSO-induced LA7 cells. Both proteins are essential in the formation of domes because when their synthesis is blocked by antisense RNA oligonucleotides, dome formation is abolished. Thus our in vitro system is a model for lobulo-alveolar development, and the genes identified in the pathway of dome formation are likely to be involved in the early differentiation steps occurring in the rat mammary gland during pregnancy and lactation.
Assuntos
Diferenciação Celular , Glândulas Mamárias Animais/citologia , Animais , Anexina A1/fisiologia , Sequência de Bases , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Primers do DNA , Dimetil Sulfóxido/farmacologia , Eletroforese em Gel Bidimensional , Proteínas de Choque Térmico HSP90/fisiologia , Hidrocortisona/farmacologia , Proteínas do Leite/genética , Prolactina/farmacologia , Proteoma , Ratos , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
We report the establishment in vitro of three-dimensional (3D) cultures of human osteoblasts (hOB) derived from normal adults and supported uniquely by the extracellular matrix (ECM) they deposit. Osteoblasts were cultured in 3D cultures in vitro for up to 120 days. The 3D cultures, examined at 25, 31, and 48 days, expressed protein markers of osteoblastic cells, namely osteonectin, collagen type I, fibronectin, osteopontin, bone sialoprotein, biglycan, and decorin. Sequentially, alkaline phosphatase (AP) and then Ca incorporation, mineralization of matrix (monitored by histochemistry and transmission electron microscopy), and finally osteocalcin expression, were detected in the 3D cultures. Ultrastructurally, morphology progressed from early to mature osteoblast and to osteocyte-like. Cells were embedded in a matrix with organized collagen type I fibers containing, increasingly with time of culture, needle-shaped crystals, often associated with matrix vesicles, characteristic of those in bone. During the culture (up to 120 days) there was an outgrowth of proliferating osteogenic cells from the 3D structure. Subcutaneous implantation in nude mice for 20 days of osteoblasts cultured in 3D culture for different lengths of time in vitro, showed progression of mineralization from the inner region of the implant outward, with peripheral cells being embedded in nonmineralized, collagen-rich matrix. The 3D implants were invaded by vessels derived from the host.
Assuntos
Técnicas de Cultura de Células/métodos , Osteoblastos/transplante , Osteoblastos/ultraestrutura , Adulto , Idoso , Fosfatase Alcalina/metabolismo , Animais , Biomarcadores , Calcificação Fisiológica/fisiologia , Cálcio/metabolismo , Diferenciação Celular , Divisão Celular , Feminino , Histocitoquímica , Humanos , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Nus , Microscopia Eletrônica , Pessoa de Meia-Idade , Transplante de Neoplasias , Osteoblastos/metabolismo , Osteogênese/fisiologiaRESUMO
Fgf4, a member of the fibroblast growth factor family, is frequently amplified in a variety of human cancers, however, its expression in neoplastic tissues is rarely detectable. This makes uncertain its involvement in tumour aetiology, although several in-vitro studies link Fgf4 overexpression to malignant transformation and metastatization of culture cells. We generated a transgenic mouse model in which the whey acidic protein (WAP) promoter directs expression of human Fgf4 to mammary tissues during late pregnancy and throughout lactation, with the purpose of studying the involvement of this growth factor in mammary tumorigenesis. Expression of the transgene was specifically detected in lobular-alveolar cells of lactating mammary glands that, by histological analysis, displayed hyperplastic areas and a disorganized structure. This was accompanied by an increased number of red blood cells and expression, in alveolar epithelial cells, of the vascular endothelial growth factor, which is absent in wild type controls. The most striking effect caused by FGF4 overexpression was on the remodelling of mammary tissue at the end of lactation. Indeed, transgenic animals showed a delayed involution of the gland due to a dramatic reduction in the overall number of apoptotic cells, which are normally present in the organ after weaning. Nevertheless, none of the animals examined developed neoplastic lesions of the mammary gland even after several pregnancies and at old age. Our work represents the first in-vivo demonstration of the anti-apoptotic and angiogenic properties of FGF4.
Assuntos
Apoptose , Fatores de Crescimento de Fibroblastos/fisiologia , Hiperplasia/patologia , Glândulas Mamárias Animais/anormalidades , Glândulas Mamárias Animais/patologia , Neovascularização Patológica , Proteínas Proto-Oncogênicas/fisiologia , Envelhecimento/fisiologia , Animais , Western Blotting , Transformação Celular Neoplásica , Fatores de Crescimento Endotelial/genética , Fatores de Crescimento Endotelial/metabolismo , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Feminino , Fator 4 de Crescimento de Fibroblastos , Fatores de Crescimento de Fibroblastos/genética , Regulação da Expressão Gênica , Humanos , Hiperplasia/sangue , Hiperplasia/genética , Hiperplasia/metabolismo , Imuno-Histoquímica , Lactação , Linfocinas/genética , Linfocinas/metabolismo , Glândulas Mamárias Animais/irrigação sanguínea , Glândulas Mamárias Animais/metabolismo , Neoplasias Mamárias Animais/irrigação sanguínea , Neoplasias Mamárias Animais/genética , Neoplasias Mamárias Animais/metabolismo , Neoplasias Mamárias Animais/patologia , Camundongos , Camundongos Transgênicos , Proteínas do Leite/análise , Proteínas do Leite/biossíntese , Proteínas do Leite/genética , Fenótipo , Gravidez , Regiões Promotoras Genéticas/genética , Proteínas Proto-Oncogênicas/genética , RNA Mensageiro/análise , RNA Mensageiro/genética , Transgenes/genética , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularRESUMO
The Dlx5 gene encodes a Distal-less-related DNA-binding homeobox protein first expressed during early embryonic development in anterior regions of the mouse embryo. In later developmental stages, it appears in the branchial arches, the otic and olfactory placodes and their derivatives, in restricted brain regions, in all extending appendages and in all developing bones. We have created a null allele of the mouse Dlx5 gene by replacing exons I and II with the E. coli lacZ gene. Heterozygous mice appear normal. Beta-galactosidase activity in Dlx5+/- embryos and newborn animals reproduces the known pattern of expression of the gene. Homozygous mutants die shortly after birth with a swollen abdomen. They present a complex phenotype characterised by craniofacial abnormalities affecting derivatives of the first four branchial arches, severe malformations of the vestibular organ, a delayed ossification of the roof of the skull and abnormal osteogenesis. No obvious defect was observed in the patterning of limbs and other appendages. The defects observed in Dlx5-/- mutant animals suggest multiple and independent roles of this gene in the patterning of the branchial arches, in the morphogenesis of the vestibular organ and in osteoblast differentiation.
Assuntos
Osso e Ossos/anormalidades , Anormalidades Craniofaciais/genética , Genes Homeobox , Proteínas de Homeodomínio/genética , Vestíbulo do Labirinto/anormalidades , Animais , Animais Recém-Nascidos , Apoptose/genética , Sequência de Bases , Encéfalo/anormalidades , Diferenciação Celular/genética , Divisão Celular/genética , Primers do DNA/genética , Regulação da Expressão Gênica no Desenvolvimento , Marcação de Genes , Hibridização In Situ , Óperon Lac , Camundongos , Camundongos Knockout , Mutação , Osteoblastos/citologia , FenótipoRESUMO
F9 embryonal carcinoma cells can differentiate in vitro into either parietal (PE) or visceral (VE) endoderm, depending upon specific retinoic acid (RA) treatment and growth conditions. In differentiated aggregates of F9 cells (EB), the VE is a polarized monolayer surrounding a core of undifferentiated cells. Within 7 days of treatment the cells organize their cytoskeleton and synthesize large amounts of extracellular matrix proteins to form a basal lamina under the newly formed epithelium. All these changes are likely to involve integrin expression and organization. In this study we have analyzed the spatio-temporal changes in the pattern and level of expression of beta1, beta4, alpha5, alpha6A, and alpha6B integrin subunits. We found that the organization of the VE monolayer in F9 aggregates involves both qualitative and quantitative changes in integrin expression. beta1 is downregulated and accumulates in the forming epithelium. The same occurs for alpha5, although its location on the surface of the aggregate appears to be transient as in fully differentiated EB its distribution is uniform. beta4 and alpha6A are also mainly localized in the VE but they are undetectable in undifferentiated aggregates and their expression is induced by RA treatment. An important exception is represented by alpha6B whose distribution and expression remain almost unchanged throughout treatment.
Assuntos
Carcinoma Embrionário/metabolismo , Integrinas/metabolismo , Teratocarcinoma/metabolismo , Animais , Carcinoma Embrionário/genética , Carcinoma Embrionário/patologia , Embrião de Mamíferos/metabolismo , Embrião de Mamíferos/patologia , Desenvolvimento Embrionário e Fetal , Endoderma/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Integrina beta1/biossíntese , Integrina beta1/genética , Integrinas/biossíntese , Laminina/biossíntese , Camundongos , Receptores de Fibronectina/biossíntese , Receptores de Laminina/biossíntese , Teratocarcinoma/genética , Teratocarcinoma/patologia , Células Tumorais CultivadasRESUMO
Objetivo: determinar la existencia de déficit en la comunicación oral de menores atendidos en un consultorio de atención primaria del Area Metropolitana. Pacientes y método: se evaluó el lenguaje en 316 niños de ambos sexos entre uno y siete años de edad, con una escala de desarrollo de la comunicación oral e instrumentos específicos para los distintos niveles del lenguaje. Resultados: el 6,8 por ciento de la población infantil presenta algún tipo de déficit del lenguaje. Se observa un predominio del sexo masculino (66,4 por ciento) y una mayor frecuencia entre los tres y cuatro años 11 meses (66,4 por ciento). Conclusión: existe un número importante de niños con trastornos de la comunicación oral que pueden ser detectados a nivel de atención primaria, por lo que sería la instancia de diagnóstico e intervención oportunos. Esta información en el ámbito de la salud permitiría tomar medidas apropiadas, como implementar programas de atención fonoaudiológica en el nivel primario
Assuntos
Humanos , Masculino , Feminino , Lactente , Pré-Escolar , Transtornos da Linguagem/diagnóstico , Distúrbios da Fala/diagnóstico , Transtornos da Linguagem/classificação , Consultórios Médicos , Distribuição por Sexo , Distúrbios da Fala/classificação , GagueiraRESUMO
Oncogene-bearing transgenic mice develop various kinds of tumors depending on both the regulatory sequences and the specific oncogene used. These mice not only help to clarify the pathogenetic pathways leading to tumor formation, but can also be useful as models to test novel therapeutic strategies, including gene therapy. We have previously reported the establishment of an MMTV-neu (ErbB-2) transgenic mouse lineage, in which 100% of females develop breast tumors with many features similar to their human counterparts; these tumors are due to the over-expression of the activated rat neu oncogene in the mammary gland. From one such mouse we established a cell line of mammary adenocarcinoma named MG1361. We report here that the growth of this cell line can be inhibited in vitro and in vivo by transfection of a plasmid vector carrying an antisense anti-neu construct. This inhibitory effect is specific, as it is related to the expression of the antisense transgene (determined by RT-PCR), and to a reduction in neu mRNA and protein, as determined by Northern and Western blot analyses. Moreover, inoculation of cells carrying the antisense or the control vector in nude mice demonstrated that the morphological and biochemical effects elicited by the antisense construct resulted in a significantly slower rate of in vivo growth of tumor xenografts. Finally, significant mammary tumor growth inhibition was obtained after liposome-mediated direct inoculation of the same antisense vector in tumors spontaneously arising in MMTV-neu mice. Taken together, these findings suggest that targeting neu expression by an integrated large anti-neu antisense segment affects the in vivo growth of these tumors.
Assuntos
Adenocarcinoma/genética , Elementos Antissenso (Genética) , Genes erbB-2 , Neoplasias Mamárias Experimentais/genética , Adenocarcinoma/patologia , Animais , Feminino , Regulação Neoplásica da Expressão Gênica , Técnicas de Transferência de Genes , Vetores Genéticos , Neoplasias Mamárias Experimentais/patologia , Vírus do Tumor Mamário do Camundongo/genética , Camundongos , Camundongos Nus , Camundongos Transgênicos , Transplante de Neoplasias , Reação em Cadeia da Polimerase , RNA Mensageiro/genética , Receptor ErbB-2/genética , Receptor ErbB-2/metabolismo , Células Tumorais CultivadasRESUMO
A new murine cell line, named MG1361, was established from mammary adenocarcinomas arising in a MMTV-neu transgenic mouse lineage where breast tumors develop in 100% of females, due to the overexpression of the activated rat neu oncogene in the mammary gland. The MG1361 cell line shows an epithelial-like morphology, has a poor plating efficiency, low clonogenic capacity, and a doubling time of 23.8 hours. Karyotype and flow cytometry analysis revealed a hypotetraploid number of chromosomes, whereas cell cycle analysis showed 31.2% of cells to be in the G1 phase, 21.4% in S and 47.4% in G2 + M. This cell line maintains a high level of neu expression in vitro. The MG1361 cell line was tumorigenic when inoculated in immunodeficient (nude) mice and the derived tumors showed the same histological features as the primary tumors from which they were isolated. MG1361 cells were positive for specific ER and PgR binding which was competed by tamoxifen, making this cell line useful for the evaluation of endocrine therapy. Moreover, they were sensitive to etoposide treatment, suggesting that they could be a model for the study of chemotherapy-induced apoptosis. As the tumors arising in MMTV-neu transgenic mice have many features in common with human mammary adenocarcinomas (Sacco et al., Gene Therapy 1995; 2: 493-497), this cell line can be utilized to perform basic studies on the role of the neu oncogene in the maintenance of the transformed phenotype, and to test novel protocols of therapeutic strategies.
Assuntos
Adenocarcinoma , Neoplasias da Mama , Animais , Técnicas de Cultura , Feminino , Citometria de Fluxo , Cariotipagem , Camundongos , Camundongos Transgênicos , Receptor ErbB-2/genética , Receptores de Estrogênio/análise , Receptores de Progesterona/análise , Células Tumorais Cultivadas/ultraestruturaRESUMO
In vitro differentiation of the murine embryonal carcinoma (EC) cell line F9 parallels that of the early blastocyst, where visceral (VE) and parietal endoderm (PE) diverge from a common precursor, the primitive endoderm. This differentiation pathway is induced by retinoic acid (RA) and dibutyryl cyclicAMP (dcAMP) and is accompanied by progressive and dramatic changes in cell morphology and functions. Within 7 days of treatment the cells organize their cytoskeleton and synthesize large amounts of extracellular matrix proteins, becoming fully differentiated migratory cells; all these changes are likely to involve integrins expression and organization. We have investigated the changes in beta 1 integrin expression, its maturation, and organization on the cell surface in association with alpha 6, during the transition from undifferentiated F9 stem cells to migrating PE cells. By Western blotting and immunoprecipitation we showed a gradual decrease in the amount of the beta 1 subunit on the cell surface and a parallel progressive accumulation of immature protein, indicating that the control of beta 1 expression during F9 cells differentiation occurs first at post-translational level and then at the level of transcription. Moreover, the induction of differentiation produces a marked decrease of alpha 6B and its association to a high molecular weight protein, while alpha 6A level increases. By immunofluorescence we found that upon differentiation there is a relocation of the beta 1 and alpha 6B integrin subunits from cell-cell contacts to focal contacts where they colocalize with vinculin. On the contrary alpha 6A, weakly present in F9 stem cells, is present in the focal contacts of PE cells and along the stress fibers. We suggest different roles for the two alpha 6 isoforms.
Assuntos
Endoderma/citologia , Integrinas/biossíntese , Células-Tronco Neoplásicas/citologia , Animais , Bucladesina/farmacologia , Compartimento Celular , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Movimento Celular , Citoesqueleto/química , Células-Tronco de Carcinoma Embrionário , Integrina alfa6beta1 , Integrina beta1/análise , Integrinas/genética , Junções Intercelulares/química , Junções Intercelulares/fisiologia , Camundongos , Organelas/química , Tretinoína/farmacologia , Células Tumorais Cultivadas , Vinculina/análiseRESUMO
Females from a mouse lineage transgenic for the activated rat neu oncogene under the control of the mouse mammary tumor virus (MMTV) long terminal repeat (LTR) all develop breast tumors with high reproducibility within the first 2-3 months of life. These animals were crossed with mice from a lineage transgenic for the herpes simplex virus thymidine kinase gene (HSVtk) under the control of its own promoter and polyoma enhancer. Double transgenic mice (for both neu and tk) developed breast neoplasias with the same kinetics as the neu-only mice. Tumor-bearing double transgenic mice, treated intratumorally with the antiviral agent ganciclovir (GCV), showed an inhibiting effect on tumor growth. However, this effect was not seen either on GCV-treated neu-only transgenic mice or on saline-injected controls. This suggests that tk-engineered breast tumors are susceptible to GCV administered locally, and implies that neu-mice could be a useful model for testing the effectiveness of HSVtk-bearing vectors followed by systemic GCV on breast cancer cells.
Assuntos
Antivirais/uso terapêutico , Ganciclovir/uso terapêutico , Genes erbB-2/genética , Neoplasias Mamárias Experimentais/tratamento farmacológico , Simplexvirus/genética , Timidina Quinase/genética , Animais , Sequência de Bases , Feminino , Expressão Gênica , Neoplasias Mamárias Experimentais/genética , Neoplasias Mamárias Experimentais/patologia , Camundongos , Camundongos Transgênicos , Dados de Sequência Molecular , Simplexvirus/enzimologiaRESUMO
During extensive investigations on the effects of oncogenic retroviruses in developing rodents, the ability of MSV to mount a neoplastic response in CD-I Swiss mouse embryos was determined. By infecting the animals directly in utero at selected stages of post-implantation development, we detected a peculiar reaction of the embryonal tissues to certain MSVs: when mice were exposed to KiMSV at mid-gestation, the newborn developed characteristic tumors, in addition to mesenchymal cell sarcomas, not induced in fetuses and neonates. These included pulmonary alveologenic tumors and skin papillomas and were seen in mice infected on days 8 and 10 of pregnancy, roughly corresponding to 15 and 35 somites, respectively. To determine the specificity of these events, other 8- and 10-day-old embryos were infected with retroviruses of the same or different families. HaMSV and MoMSV also induced mesenchymomas and a low incidence of skin papillomas (10% and 15% compared to 40% in the KiMSV group) but not pulmonary tumors. In contrast, FBRMSV was inactive in this respect and only osteogenic sarcomas were detected in the offspring. Infecting the embryos on day 7 of pregnancy produced no tumors. Later infections (in 15-day-old fetuses and neonates) mainly induced mesenchymal sarcomas. No congenital malformations were detected in the embryos exposed to MSV during organogenesis, although some abortions and resorptions were seen.
Assuntos
Papiloma/microbiologia , Sarcoma Experimental/microbiologia , Neoplasias Cutâneas/microbiologia , Animais , Feminino , Neoplasias Pulmonares/microbiologia , Troca Materno-Fetal , Camundongos , Osteossarcoma/microbiologia , Gravidez , Vírus do Sarcoma MurinoAssuntos
Fibrilação Atrial/tratamento farmacológico , Propafenona/administração & dosagem , Taquicardia Supraventricular/tratamento farmacológico , Síndrome de Wolff-Parkinson-White/complicações , Adulto , Fibrilação Atrial/diagnóstico , Eletrocardiografia , Feminino , Humanos , Infusões Intravenosas , Taquicardia Supraventricular/diagnósticoRESUMO
We have studied the teratogenicity of benzo(a)pyrene (BP), benzo(a)pyrene-4,5-oxide, and a racemic mixture of 7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene, a proximal metabolite and ultimate carcinogenic metabolite of BP, respectively, and of 6-methylbenzo(a)pyrene after direct injection into embryonal Swiss mice. The compounds were dissolved in acetone and trioctanoin (1:1) and injected at doses ranging from 0.4 to 16.0 nmol/embryo on days 10, 12, and 14 of development. The transplacental effects of BP given at the same gestational days and at comparable dose levels were also evaluated. The control groups received 0.5, 1.0, or 2.0 microliter/embryo of vehicle on days 10, 12, or 14 of pregnancy, respectively. The fetuses were examined when they were 18 days old. On the basis of gross external and internal malformations, 7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene appeared to be the most potent embryotoxic and teratogenic compound tested, causing 85% of embryolethality and 100% of malformed fetuses in the group treated on day 10 of intrauterine development. There were 61 and 27% of malformed fetuses following 7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene treatment on days 12 and 14 of gestation, respectively. The effects of this BP metabolite were very specific and malformations such as exencephaly, thoraco- and gastroschisis, phocomelia, and edema were found. The administration of BP (both transplacental and direct intraembryonal injection) and benzo(a)pyrene-4,5-oxide caused no significant increase of malformed fetuses in any of the developmental stages considered. 6-Methylbenzo(a)pyrene induced multiple malformations (among these a high percentage of protruding tongue) in 50, 46 and 31% of the fetuses treated on days 10, 12, and 14 of gestational age, respectively. These results combined with previous data concerning the induction of lung tumors by the tested compounds in 15-day-old Swiss mouse embryos, emphasize the requirement of a common metabolic derivative of BP to induce both teratogenesis and carcinogenesis in mice. Furthermore present data show that midgestation Swiss embryos are also highly sensitive to the 6-methyl derivative of BP.
Assuntos
Anormalidades Induzidas por Medicamentos/etiologia , Benzo(a)pireno/toxicidade , Benzopirenos/toxicidade , Animais , Benzopirenos/síntese química , Relação Dose-Resposta a Droga , Feminino , Morte Fetal/induzido quimicamente , Idade Gestacional , Camundongos , Placenta/efeitos dos fármacos , Gravidez , Relação Estrutura-AtividadeRESUMO
The Moloney (MoMSV) and Kirsten (KiMSV) strains of murine sarcoma viruses are known to induce mesenchymal sarcomas upon infection of newborn rodents. To determine their activity in mouse embryos, 11- to 15-day-pregnant CD-1 mice were laparotomized, and the single implants were inoculated into the abdominal portion of the embryonal body with an average of 15 and 1500 focus-forming particles/g of body weight of the MoMSV and KiMSV viruses, respectively. Another group of less than 1-day-old pups was given a comparable amount of either virus. Tumors appeared in the young within the first few weeks of life with incidences and histological types dependent on the gestational day and the viral strain inoculated. Mixed mesenchymal sarcomas at or near the site of inoculation and vascular tumors of the brain were by far the most frequent neoplasms observed in the newborn. With MoMSV there was an increased incidence of sarcomas with advancing age at treatment, being 0% at 11 days of pregnancy and 96% in newborn (P for trend, less than 0.025). By contrast, KiMSV caused an incidence of sarcomas below 20% throughout (P for trend, greater than 0.05). Brain tumors were identified in the several MoMSV and KiMSV groups, with a peak value of 43% following the inoculation of both viruses into 13- and 15-day-old embryos, respectively. While the total incidence of these tumors was significantly different from controls, no positive trend by day of treatment was found among the MoMSV and KiMSV viruses (P less than 0.05). The tumors were mainly capillary angiomas, but a few cavernous angiomas were also detected. In addition, eight pups which were given injections of both viruses at developmental Days 11 to 13 had tumors of the choroid plexus. In many instances, newborn pups were affected by multiple vascular abnormalities of the brain, including capillary telangiectases and multiple hemorrhagic areas. No such lesions nor tumors at any site were found among the control animals. The present results are important not only because of the evidence that Swiss embryos respond selectively to the carcinogenic effects by murine sarcoma viruses, but also because they offer the opportunity to dissect directly in vivo the mechanisms underlying the stage-related sensitivity of prenatal mice to oncogenic retroviruses.